Hi I am trouble shooting for the standard curves and would appreciate any input. I am using supermix (Biorad) in iCycler iQ (Biorad) machine. When I used the same primer/template/setting around one year ago, the PCR efficiency of the standard curve is around 100%. However, when I used the same reagents this month, PCR efficiency is usually around 65-75%. I have tested the following: 1) different batches of supermix from my lab and other labs, 2) re-maxiprep plasmid template, 3) tested primers from different batch but none of them had any effect. The weird thing is when cDNA dilutions were used to establish the standard curve, the PCR efficiency is close to 100%. Due to the nature of absolute quantification, I have to use plasmid as the template. I am wondering if you have any suggestions on how to improve PCR efficiency. Thanks in advance!

-aphroditeH-

aphroditeH on Aug 19 2009, 05:19 PM said:

Hi I am trouble shooting for the standard curves and would appreciate any input. I am using supermix (Biorad) in iCycler iQ (Biorad) machine. When I used the same primer/template/setting around one year ago, the PCR efficiency of the standard curve is around 100%. However, when I used the same reagents this month, PCR efficiency is usually around 65-75%. I have tested the following: 1) different batches of supermix from my lab and other labs, 2) re-maxiprep plasmid template, 3) tested primers from different batch but none of them had any effect. The weird thing is when cDNA dilutions were used to establish the standard curve, the PCR efficiency is close to 100%. Due to the nature of absolute quantification, I have to use plasmid as the template. I am wondering if you have any suggestions on how to improve PCR efficiency. Thanks in advance!

Seems something in the template for making standard curve inhibit the pcr amplification, that is why when you diluted, the efficiency is OK. I would suggest prepare a new batch plasmid, make sure free of RNA, protein and endotoxin. Good luck.

-frankfan-

Are you linearising your plasmid? I wasn't sure from what you wrote - I have read that not doing this can affect the efficiency

-front_hands-

frankfan on Sep 4 2009, 03:40 PM said:

aphroditeH on Aug 19 2009, 05:19 PM said:

Hi I am trouble shooting for the standard curves and would appreciate any input. I am using supermix (Biorad) in iCycler iQ (Biorad) machine. When I used the same primer/template/setting around one year ago, the PCR efficiency of the standard curve is around 100%. However, when I used the same reagents this month, PCR efficiency is usually around 65-75%. I have tested the following: 1) different batches of supermix from my lab and other labs, 2) re-maxiprep plasmid template, 3) tested primers from different batch but none of them had any effect. The weird thing is when cDNA dilutions were used to establish the standard curve, the PCR efficiency is close to 100%. Due to the nature of absolute quantification, I have to use plasmid as the template. I am wondering if you have any suggestions on how to improve PCR efficiency. Thanks in advance!

Seems something in the template for making standard curve inhibit the pcr amplification, that is why when you diluted, the efficiency is OK. I would suggest prepare a new batch plasmid, make sure free of RNA, protein and endotoxin. Good luck.

Thanks for the reply. I do suspect that's the case. However, dilutions of plasmid template still have low pcr efficiency, even though dilutions of cDNA work perfectly. I did try to prepare a new batch plasmid but no difference.

-aphroditeH-

front_hands on Sep 8 2009, 10:39 AM said:

Are you linearising your plasmid? I wasn't sure from what you wrote - I have read that not doing this can affect the efficiency

Hi! I did not linearize plasmids before. I will give it a try this time. Thanks!

-aphroditeH-

aphroditeH on Sep 8 2009, 12:33 PM said:

front_hands on Sep 8 2009, 10:39 AM said:

Are you linearising your plasmid? I wasn't sure from what you wrote - I have read that not doing this can affect the efficiency

Hi! I did not linearize plasmids before. I will give it a try this time. Thanks!

I tested linearlized plasmids versus uncut ones but there is no difference.

Any other ideas why the pcr amplifies at high efficiency (>90%) when dilutions of cDNA was used as the template but at low efficiency (70%) when plasmids were used. Thanks!